The discovery of catalytically active RNA has provided the basis for the evolutionary concept of an RNA world. It has been proposed that during evolution the functions of ancient catalytic RNA were modulated by low molecular weight effectors, related to antibiotics, present in the primordial soup. Antibiotics and RNA may have co-evolved in the formation of the modern ribosome (Davies, J. Molec. Microbio. 4, 1227-1232 (1990)). The experiments described herein demonstrate that a set of aminoglycoside antibiotics, which are known to interact with the decoding region of the 16S ribosomal RNA of Escherichia coli. (Moazed, D. & Noller, H. F. Nature 327, 389-394 (1987); Noller, H. F. A. Rev. Biochem. 53, 119-162 (1984); Cundliffe, E. in The Ribosome (eds Hill, W. E. et al.) 479-490 (Am. Soc. Microbiol., Washington 1990), inhibit the second step of splicing of the T4 phage-derived td intron. Thus catalytic RNA seems to interact not only with a mononucleotide (Bass, B. & Cech, T. R. Biochemistry 25, 44773-4477 (1986)) and an amino acid (Yarus, M. Science 240, 1751-1758 (1988)), but also with another class of biomolecules, the sugars. Splicing of other group I introns but not group II introns was inhibited. The similarity in affinity and specificity of these antibiotics for group I introns and rRNAs may result from recognition of evolutionarily conserved structures.
The first step of splicing, which is initiated by binding of exogenous guanosine to the G-binding site, is dependent on guanosine concentration. Once bound to the ribozyme, the 5' splice-site is cleaved and the nucleoside becomes covalently linked to the first nucleotide of the intron. The second step of splicing is initiated by nucleophilic attack of the 3' splice site by the free 3' hydroxyl group of the upstream exon. The splice site is cleaved and the exons are ligated (FIG. 1). (Cech, T. R. A. Rev. Biochem. 59, 543-568 (1990).) The genetic information carried by DNA is transcribed to RNA to mediate the process of gene expression. The coding region of RNA is very often interrupted by so called intervening sequences (introns). These introns must be removed from the coding regions (exons) to result in an uninterrupted sequence or functional RNA; the exons become spliced together. Depending on the splice mechanism, characteristic secondary structure, and cofactor dependence, several intron classes can be differentiated. The group I intron RNA can be folded in a characteristic set of stem and loop structure. These introns are removed by two consecutive transesterification reactions and an external guanosine or guanosine phosphate derivative is needed to initiate the splice reaction. This guanosine attacks, via its 3'-hydroxyl group, the 5'splice site and becomes covalently linked to the 5'end of the intron. The 3'-hydroxyl of the 5'exon now attacks the 3' splice site, the two exons become ligated and the intron is released. This reaction is autocatalytic, i.e., the reaction occurs in the absence of any protein enzymes or hydrolysis of energy rich bonds solely because of the ability of the intron RNA to fold in its active form. The reaction can be performed in vitro by synthesizing the RNA in vitro and incubating it in the presence of pH-buffer, magnesium ions and the cofactor guanosine (or GTP) (see Cech, Annual Review of Biochemistry, 1990, vol. 59, p. 543).
Group I introns have been found in messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA) in microorganisms and organelles of all kingdoms (compare Michel & Westhof, Journal of Molecular Biology, 1990, vol. 216, p.585).
The group I intron provides an attractive target for antimicrobial agents because the intron is predominantly found in lower eukaryotes. The invention described herein provides methods for screening for compounds that inhibit group I intron splicing and methods for inhibiting microbial growth with these compounds.